Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field

The reduced mycorrhizal colonization (\u3ci\u3ermc\u3c/i\u3e) mutation of tomato disrupts five gene sequences including the \u3ci\u3eCYCLOPS/IPD3\u3c/i\u3e homologue

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signaling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonization (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterization of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterization of rmc in comparison with its ‘parent’ 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots. Includes supplementary material

Leptosphaeria maculans Effector Protein AvrLm1 Modulates Plant Immunity by Enhancing MAP Kinase 9 Phosphorylation

Summary: Leptosphaeria maculans, the causal agent of blackleg disease in canola (Brassica napus), secretes an array of effectors into the host to overcome host defense. Here we present evidence that the L. maculans effector protein AvrLm1 functions as a virulence factor by interacting with the B. napus mitogen-activated protein (MAP) kinase 9 (BnMPK9), resulting in increased accumulation and enhanced phosphorylation of the host protein. Transient expression of BnMPK9 in Nicotiana benthamiana induces cell death, and this phenotype is enhanced in the presence of AvrLm1, suggesting that induction of cell death due to enhanced accumulation and phosphorylation of BnMPK9 by AvrLm1 supports the initiation of necrotrophic phase of L. maculans infection. Stable expression of BnMPK9 in B. napus perturbs hormone signaling, notably salicylic acid response genes, to facilitate L. maculans infection. Our findings provide evidence that a MAP kinase is directly targeted by a fungal effector to modulate plant immunity. : Parasitology; Plant Biology; Interaction of Plants with Organisms Subject Areas: Parasitology, Plant Biology, Interaction of Plants with Organism

Temperature affects the effectiveness of R gene mediated resistance against Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape)

Poster abstractPhoma stem canker (Leptosphaeria maculans) is an economically important disease on oilseed rape (Brassica napus) in Europe, Australia and North America. Effective control of this disease relies on the use of two types of resistance: major resistance (R) gene-mediated qualitative resistance and quantitative resistance (QR). Plant–pathogen interactions are known to be affected by environmental factors, including temperature. To investigate effects of temperature on durability of R gene-mediated resistance to L. maculans, oilseed rape cultivars or breeding lines with different R genes in backgrounds with/without QR were inoculated at 20°C and 25°C. Cotyledons of 12-day old plants were wounded and a drop of 10µl of 107 spores/ ml conidial suspension placed over the wound. There were differences in temperature sensitivity between the ten different R genes (Rlm1, Rlm2, Rlm3, Rlm4, Rlm5, Rlm6, Rlm7, LepR1, LepR2 and LepR3) tested and there were differences in response to temperature for the same R gene in different cultivars. Background QR affected the temperature sensitivity of R genemediated resistance. To avoid the effects of background QR and investigate plant defence responses, near isogenic lines of Topas with R genes Rlm4 or LepR3 were used. Cotyledons of 12-day old plants were infiltrated with 10 µl of 106 spore/ml conidial suspension at 20°C and 25°C. There were differences in defence responses between Rlm4 and LepR3, with Rlm4 responding more quickly and more strongly than LepR3 at the higher temperature. Understanding effects of temperature on interactions between hosts and pathogens will help to breed cultivars with durable, temperature-resilient resistanceNon peer reviewe

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L.maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B.napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates

Proportions of <i>Brassica napus</i> genes encoding receptor-like proteins (RLPs), secreted peptides (SPs) or nucleotide-binding leucine-rich repeat receptors (NLRs) within chromosomal intervals (loci) for resistance against <i>Sclerotinia sclero</i>tiorum (necrotroph), <i>Leptosphaeria maculans</i> (extracellular) or <i>Plasmodiophora brassicae</i> (intracellular).

<p>(A) Proportions represent the numbers of RLP, SP or NLR genes within the mapped regions for resistance against <i>S</i>. <i>sclero</i>tiorum, <i>L</i>. <i>maculans</i> or <i>P</i>. <i>brassicae</i> divided by the total number of genes for each of the three families. Contingency tests show that proportions of RLP, SP and NLR genes are not equal for resistance against <i>P</i>. <i>brassicae</i> (<i>χ</i>^<i>2</i> = 9.84, <i>P</i> = 0.007). (B) Proportions represent the total number of LRR genes (RLP, SP and NLR) within mapped regions for resistance divided by the total number of LRR genes (black bars). These proportions were compared to those for all of the genes within mapped regions for resistance divided by the total number of genes within the genome (white bars). Differences between these two proportions were not significant (contingency table, <i>χ</i>^<i>2</i> = 1.90, <i>P</i> = 0.39).</p

Test for evidence of positive selection in coding sequences of <i>Brassica napus R</i> genes and candidate genes for resistance against <i>Leptosphaeria maculans</i>.

<p>Test for evidence of positive selection in coding sequences of <i>Brassica napus R</i> genes and candidate genes for resistance against <i>Leptosphaeria maculans</i>.</p

Symptoms and expression of candidate <i>R</i> genes after inoculation of susceptible <i>Brassica napus</i> cultivar Topas DH16516 with <i>Leptosphaeria maculans</i> isolate 00–100.

<p>(A) Appearance of cotyledons 0, 2, 4, 6 and 8 days post-inoculation (dpi) with <i>L</i>. <i>maculans</i>. (B) Heat maps of differentially expressed genes (DEG) encoding receptor-like proteins (RLPs), secreted peptides (SPs) and nucleotide-binding leucine-rich repeat receptors (NLRs). The expression of each gene is based on regularized logarithmic transformation (rld) of the average of the best three biological replicates. Expression patterns are grouped into initial endophytic (0–2 dpi), intermediate (4 dpi) and late necrotrophic (6–8 dpi) stages of colonization [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198201#pone.0198201.ref022" target="_blank">22</a>]. (C, D) Classification of different gene families into different expression categories. Circles (from inside to outside) represent NLR, RLP and SP genes. (C) General expression patterns, reads per kilobase million (RPKM), did not differ between RLP, SP and NLR genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198201#pone.0198201.s004" target="_blank">S4 Table</a>, <i>χ</i>^<i>2</i> = 2.51, <i>P</i> = 0.64). (D) Percentages of differentially expressed genes (% DEG) at the three stages of colonization are shown. Induced expression patterns differed between RLP, SP and NLR genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198201#pone.0198201.s005" target="_blank">S5 Table</a>, <i>χ</i>^<i>2</i> = 22.13, <i>P</i> = 0.0002).</p

Distribution of candidate genes for resistance against pathogens in the genome of <i>Brassica napus</i>.

<p>All 19 (10 A and 9 C) chromosomes are displayed in grey as concentric circles according to the published <i>B</i>. <i>napus</i> genome sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198201#pone.0198201.ref021" target="_blank">21</a>]. Genes encoding nucleotide-binding leucine-rich repeat receptors (NLRs), receptor-like proteins (RLPs) and secreted peptides (SPs) are color-coded in orange, blue and red, respectively. The center of this diagram shows homeologous relationships on the A and C sub-genomes between orthologous RLP and SP gene pairs in blue and red, respectively.</p